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In Vitro Conservation of Turbinicarpus (Cactaceae) Under Slow

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 In Vitro Conservation of Turbinicarpus (Cactaceae) Under Slow
Growth Conditions
Author(s): Eugenio Pérez-Molphe-Balch, Martha Evelia Pérez-Reyes, MA. De
Lourdes De La Rosa-Carrillo
Source: Haseltonia, 17():51-57. 2012.
Published By: Cactus and Succulent Society of America
DOI: http://dx.doi.org/10.2985/1070-0048-17.1.6
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Haseltonia 17: 51–57. 2012 51

IN VITRO CONSERVATION OF TURBINICARPUS (CACTACEAE) UNDER SLOW

GROWTH CONDITIONS
EUGENIO PÉREZ-MOLPHE-BALCH1
MARTHA EVELIA PÉREZ-REYES
MA. DE LOURDES DE LA ROSA-CARRILLO
Unidad de Biotecnología Vegetal, Departamento de Química,
Universidad Autónoma de Aguascalientes.
Av. Universidad 940, 20131
Aguascalientes, Ags. México
Absctract: The current work describes an in vitro conservation technique employing slow growth conditions in 16
threatened species and subspecies of the genus Turbinicarpus (Cactaceae), all native to the Chihuahuan Desert in
Mexico. It was demonstrated that the addition of osmotic agents like mannitol (30 g L-1) and sorbitol (30 g L-1)
to the culture medium, as well as low-temperature incubation (4 ± 0.5 °C), are able to reduce the in vitro growth
rate without affecting the viability of shoots subjected to these treatments. The material that was kept under the
aforementioned treatment conditions for 12 months was able to regenerate shoots through areole activation when
transferred to media containing cytokinins. These shoots are able to root, and the generated plants can adapt
and survive in soil, with similar efficiencies to those that were not subjected to slow-growth treatment. With this
methodology it is possible to maintain a bank of viable tissues of these species in vitro, with minimal maintenance
and the possibility of obtaining complete plants whenever required.

Keywords: areole activation, Cactaceae, in vitro germplasm conservation, slow-growth storage.

easy to maintain large collections in controlled en-

Introduction
Cacti are one of the most notable elements of the
floras of arid and semiarid regions in America, Mexi-
co being the country with the largest number of spe-
cies belonging to the family Cactaceae. These plants
have been used since ancient times as a source of
food, medicine and building materials. Moreover,
their use as ornamental plants and role as collec-
tion specimens is becoming increasingly important,
for which reason the demand for specimens of rare
or little-known species has grown considerably. Ac-
cordingly, cacti represent a valuable natural resource
for the regions they inhabit. Unfortunately, irratio-
nal exploitation—in most cases illegal—and habitat
destruction have caused 276 species and subspecies
of Mexican cacti to be currently considered as threat-
ened according to the laws of Mexico (Norma Ofi-
cial Mexicana NOM-059-SEMARNAT-2010). In
the international context, 65 species are listed in
the IUCN Red Book and 41 genera are included in
Appendix II of CITES (Guzmán et al. 2007). The
genus Turbinicarpus, endemic to the Mexican por-
tion of the Chihuahuan Desert, is perhaps the most
threatened among the members of the Cactaceae.
This includes about 24 species and subspecies of
small-sized plants with a very limited distribution in
the states of Coahuila, Guanajuato, Hidalgo, Queré-
taro, Nuevo León, San Luis Potosí, Tamaulipas and
Zacatecas. Turbinicarpus species are prized by collec-
tors because of their beauty and the fact that their
small size (about 5 cm in their adult stage) makes it
vironments. This has caused the drastic decline of
many natural populations due to poaching, which
has put these species in imminent danger of being
eradicated from their natural habitat (Anderson
2001; Santos-Díaz et al. 2010). An additional prob-
lem presented by this group of plants is the low ef-
ficiency of their natural propagation systems. They
do not show vegetative reproduction and their seed
production is very low. Moreover, in some species of
the genus, the germination rate reaches only about
8%. This is due both to low seed viability and to the
existence of various dormancy mechanisms (Flores et
al. 2005; Flores et al. 2008).
In situ conservation is undoubtedly the most de-
sirable way to protect endangered cacti, because it
ensures the survival of entire ecosystems. However,
in cases as serious as that of the genus Turbinicar-
pus, this strategy must be complemented with ex situ
conservation in order to ensure the survival of these
species and to make rational use of this resource in
the future. In this sense, in vitro culture could serve
as a crucial tool for the conservation and sustain-
able management of endangered cacti. Several pub-
lished studies indicate that mass in vitro propagation
is very efficient in this family, going far beyond any
conventional propagation system (Hubstenberger et
al. 1992; Santos-Díaz et al. 2010). Plants generated
in this way can be used for various purposes, thus
reducing the pressure on wild populations caused
by poaching. Furthermore, when the relevant eco-
logical studies are conducted and the conservation of
genetic diversity is ensured, the plants propagated in

1
Corresponding author E-mail: eperezmb@correo.uaa.mx
52 PÉREZ-MOLPHE-BALCH ET AL—IN VITRO CONSERVATION

vitro may be an adequate option for the restoration reports in this regard for the cedar (Renau-Morata
of decimated or eradicated wild populations. Reports et al. 2006) and the carnivorous plant Drosophyl-
on in vitro mass propagation systems include some lum lusitanicum (Goncalves & Romano 2007). Ac-
species of the genus Turbinicarpus. These studies cording to Pence (2011), in vitro mass propagation
show that it is possible to produce large quantities and germplasm conservation are the most suitable
of plants in a short time using routes such as organo- techniques for endangered species that show a low
genesis and areole activation, which is an impor- production of viable seeds and are difficult to prop-
tant contribution to the conservation of these spe- agate vegetatively. This is the case with cacti of the
cies (Mata-Rosas et al. 2001; Dávila-Figueroa et al. genus Turbinicarpus. Despite this, there is no previ-
2005). Another application of in vitro culture that ous work related to slow-growth tissue storage in
could turn out to be interesting in the case of cacti cacti, nor on the regeneration of whole plants start-
is the preservation of living tissues to form germ- ing from it. This paper describes an in vitro conser-
plasm banks. However, the maintenance of viable vation technique under slow-growth conditions used
cultures for long periods of time requires frequent in 16 endangered species and subspecies of the genus
subculturing, which generates a considerable cost in Turbinicarpus.
terms of working time and culture media, in addi-
tion to higher contamination risks related to culture Materials and Methods
manipulation. In order to avoid these disadvantages,
slow-growth systems have been described, which aim Plant material: We used shoots generated in
to extend the time between subcultures in order to vitro from the following species and subspecies: T.
facilitate and make less expensive the maintenance hoferi Lüthy & A.B. Lau, T. laui Glass & R. Foster,
of viable cultures for long periods of time. In order T. lophophoroides (Werdermann) Buxbaum, T. pseu-
to decrease the in vitro tissue growth rate, without domacrochele subsp. lausseri (Diers & L. Gerhart
affecting viability, a controlled osmotic stress can be Frank) Glass, T. pseudomacrochele subsp. pseudomac-
applied using chemical compounds such as mannitol rochele (Backeb.) Buxb. & Backeb., T. pseudopecti-
or sorbitol in the culture medium in concentrations natus (Backeberg) Glass & R. Foster, T. saueri subsp.
from 20 to 40 g L-1, which reduce the osmotic po- nelissae Halda & Panarotto, T. schmiedickeanus
tential without altering the biochemical balance of subsp. bonatzii (G. Frank) Panar., T. schmiedickeanus
plant cells. The growth rate can also be reduced by subsp. flaviflorus (G. Frank & A. B. Lau) Glass, T.
incubating the cultures at low temperatures, or by schmiedickeanus subsp. gracilis (Glass & R. A. Foster)
using growth inhibitors in the medium. Finally, a Glass, T. schmiedickeanus subsp. klinkerianus (Backe-
combination of two or more of the aforementioned berg & H. Jacobsen) N.P. Taylor, T. schmiedickea-
treatments can be an efficient alternative in order to nus subsp. macrochele (Werderm.) N.P. Taylor, T.
maintain viable tissues in slow-growth conditions schmiedickeanus subsp. schmiedickeanus (Boedeker)
(Benson 1999; Sarasan et al. 2006). This germplasm Buxbaum & Backeberg, T. schmiedickeanus subsp.
in vitro conservation technique has proven to be schwarzii (Shurly) N.P. Taylor, T. subterraneus
the most appropriate for various cultured species; (Backeberg) A.D. Zimmerman and T. valdezianus (H.
in some of these cases it is already routinely used Moeller) Glass & R. Foster. This plant material was
and its efficiency is fully tested (Engelmann 2011). taken from the in vitro mass propagation program of
Some of the species propagated under this conserva- these species that is being developed in the Unidad
tion-of-germplasm scheme are the potato (Westcott de Biotecnología Vegetal of the Universidad Autóno-
1981), coffee (Dussert et al. 1997), apple (Negri et al. ma de Aguascalientes, México (Dávila-Figueroa et al.
2000), asparagus (Bekheet 2000), enset (Negash et al. 2005).
2001) and sugarcane (Watt et al. 2009). The tissues Effect of osmotic agents and low temperature
that are kept under these conditions are usually api- incubation on in vitro growth rate: For these ex-
cal meristems or nodal segments, although somatic periments, in vitro generated shoots (10 to 15 mm
embryos have also been used (Watt et al. 2009). Ob- in height) were taken from all species and subspecies
viously, for this conservation technique to be appli- listed above. The culture medium used in all cases
cable, the stored tissues must maintain their ability was Murashige and Skoog (1962) (MS), pH 5.7 and
to regenerate complete plants once they are removed supplemented with 30 g L -1 sucrose and 10 g L-1 agar
from the slow-growth system. This has been demon- as a gelling agent. The culture flasks containing 30
strated in all of the aforementioned species. In the mL of medium were sterilized in an autoclave at 121
apple it was also found that these storage conditions °C for 15 min. Once inoculated with the shoots, the
do not cause genetic damage in the stored samples culture flasks were sealed with five layers of PVC film
(Hao & Deng 2003), and in the case of cedar, DNA wrap (Vitafilm®) and kept under continuous light
methylation patterns are not affected (Renau-Morata (54 μM m-2 s-1) at 25 ± 2 °C for all treatments ex-
et al. 2006.) cept for the low-temperature treatment. For the two
As can be seen, slow-growth systems are a rela- test treatments using different osmotic agents, man-
tively simple and low cost alternative for the conser- nitol (30 g L-1) or sorbitol (30 g L-1) was added to
vation of plant germplasm. However, despite the ad- the culture medium, and the cultures were incubated
vantages of this methodology, it has been developed at the normal temperature of 25 ± 0.2 °C. For the
very little for endangered wildlife species; there are treatment testing low-temperature, cultures were in-
 HASELTONIA VOL. 17. 2012 53

1900 4600 350 900

Control 325
1700 4100 800
Mannitol 300
1500 3600 700
Sorbitol 275
1300 3100
Relative growth

250 600
4°C
1100 2600
225 500
900 2100
200 400
700 1600
175
300
500 1100 150
300 600 125 200

100 100 100 100

a 0 15 30 45 60 b 0 15 30 45 60 c 0 15 30 45 60 d 0 15 30 45 60

325 500 450 240

300 450 220

400
275 400
200
350
250 350
Relative growth

180
225 300 300
160
200 250 250
140
175 200
200
150 120
150
150 100
125 100

100 50 100 80
e 0 15 30 45 60 f 0 15 30 45 60 g0 15 30 45 60 h 0 15 30 45 60

1800 200 430 1800

1600 Control 400

1600
Mannitol 180 370
1400 1400
Sorbitol 340
1200 160 310 1200
Relative growth

4 °C
1000 280
1000
140 250
800 800
220
600 120 190 600

400 160
400
100 130
200 200
100
0 80 70 0
i 0 15 30 45 60 j 0 15 30 45 60 k0 15 30 45 60 l 0 15 30 45 60

450 200 500 1100

450 1000
400 180
900
400
350 800
160
Relative growth

350 700
300
140 300 600
250
250 500
120
200 400
200
300
150 100
150 200
100 80 100 100
m0 15 30 45 60 n 0 15 30 45 60 o 0 15 30 45 60 p 0 15 30 45 60

Figure 1. Effect of osmotic agents and low temperature during incubation on in vitro growth rate (average ± standard error)
measured at 0, 15, 30 45 and 60 d, in: a) Turbinicarpus schmiedickeanus subsp. bonatzii; b) Turbinicarpus hoferi; c) Turbinicar-
pus laui; d) Turbinicarpus lophophoroides; e) Turbinicarpus pseudomacrochele subsp. lausseri; f ) Turbinicarpus pseudomacrochele
subsp. pseudomacrochele; g) Turbinicarpus pseudopectinatus; h) Turbinicarpus schmiedickeanus subsp. flaviflorus; i) Turbinicarpus
schmiedickeanus subsp. gracilis; j) Turbinicarpus schmiedickeanus subsp. klinkerianus; k) Turbinicarpus schmiedickeanus subsp.
macrochele; l) Turbinicarpus schmiedickeanus subsp. schmiedickeanus; m) Turbinicarpus schmiedickeanus subsp. schwarzii; n)
Turbinicarpus subterraneus; o) Turbinicarpus valdezianus; p) Turbinicarpus saueri subsp. nelissae.

cubated with the normal medium at 4 ± 0.5 °C in a three treatments (mannitol at normal temperature,
refrigerator equipped with fluorescent lights adjusted sorbitol at normal temperature, and low temperature
to an intensity of 54 μM m-2 s-1. The effects of the with no osmotic agent) on the in vitro growth rate of
54 PÉREZ-MOLPHE-BALCH ET AL—IN VITRO CONSERVATION

a b c
Figure 2

d e f
Figure 2. a) Turbinicarpus pseudopectinatus shoots after 12 months in a culture medium with mannitol; b) Turbinicarpus
schmiedickeanus subsp. klinkerianus shoots after 12 months in a culture medium with sorbitol; c) Turbinicarpus laui shoots after
12 months in a culture medium with mannitol. Production of new shoots by areole activation in explants taken from shoots
conserved for 12 months in medium with mannitol: d) T. schmiedickeanus subsp. schmiedickeanus; e) Turbinicarpus subterra-
neus; f ) Turbinicarpus laui. (bar = 10 mm). Photos: E. Pérez-Molphe. .

the shoots was determined as follows: We took the
initial fresh weight of the shoots and then weighed
them after 15, 30, 45 and 60 d of incubation. Due
to differences in the initial shoot weight within and
among species (which makes the comparison of re-
sults difficult), the relative growth was determined
considering the initial weight as 100%. For each of
the taxa tested, 15 shoots were used per treatment,
including the control treatment. Two shoots were
placed in each culture vessel. The entire experiment
was performed three or four times, depending on the
availability of plant material of each species.
Effect of in vitro conservation under slow-
growth conditions on the regeneration capacity
of cultured shoots: Shoots subjected to the afore-
mentioned treatments were incubated for a total of
12 months under the same conditions. In the case of
the control treatment, the tissues were subcultured
to fresh medium every 60 d for the same period. Tis-
sues subjected to the other three treatments were not
subcultured. After 12 months, the shoots were col-
lected, the apex and the basal portion were removed,
and the remaining tissue was cut transversely to
form slices about 4 mm thick containing one or two
rows of areoles, this depending on the species. These
transverse segments were inoculated with the wound
in contact with the medium, maintaining their origi-
nal polarity, in MS medium (pH 5.7), supplemented
with 30 g L-1 sucrose, 10 g L-1 agar and 1 mg L-1
benzylaminopurine (BA), the latter included to in-
duce areole sprouting, the first step in whole plant
regeneration. A total of 20 explants from each test
treatment and the control were inoculated for each
taxon. The entire experiment was performed twice.
The cultures were kept under continuous light (54
μM m-2 s-1) at 25 ± 2 °C for 60 d. After this time, the
number of shoots produced per explant was recorded.
The data so obtained were analyzed by ANOVA and
the comparison between the mean values was done
using a Tukey test with values of P ≤ 0.05 consid-
ered significant. This was done in order to determine
whether the slow-growth systems affected the regen-
eration capacity of tissues stored for 12 months. The
shoots generated in these experiments were separated
from the original explant and transferred to 50% MS
medium at pH 5.7, supplemented with 15 g L-1 su-
crose and 10 g L-1 agar. They were incubated for 45
d at 25 ± 2 °C under continuous light (54 μM m-2
s-1) in order to induce rooting. The generated plants
were adapted and transferred to soil using a previ-
ously developed methodology (Dávila-Figueroa et al.
2005).
Results and Discussion
We worked with shoots from 16 species and sub-
species of the genus Turbinicarpus; it was observed
that their growth rate in vitro in a basal medium
 HASELTONIA VOL. 17. 2012 55

Treatment (shoots per explant*)

Turbinicarpus species Control Mannitol Sorbitol 4 °C
30 g L-1 30 g L-1
T. hoferi 2.5 a 2.1 a 2.1 a 2.3 a
T. laui 4.0 a 4.5 a 2.6 a 5.5 a
T. lophophoroides 2.9 a 4.0 a 3.9 a 2.9 a
T. pseudomacrochele 13.7 a 13.6 a 13.1 a 13.7 a
subsp. lausseri
T. pseudomacrochele 3.7 a 3.3 ab 4.0 a 1.9 b
subsp. pseudomacrochele
T. pseudopectinatus 23.1 a 18.2 b 21.7 ab 23.9 a
T. saueri subsp. nelissae 3.4 a 3.1 a 2.9 a 3.8 a
T. schmiedickeanus 6.3 ab 7.7 a 5.9 ab 4.7 b
subsp. bonatzii
T. schmiedickeanus 4.5 a 4.3 a 5.3 a 4.1 a
subsp. flaviflorus
T. schmiedickeanus 6.2 a 5.9 a 5.1 a 5.1 a
subsp. gracilis
T. schmiedickeanus 2.1 a 2.1 a 3.7 a 4.5 a
subsp. klinkerianus
T. schmiedickeanus 2.4 ab 2.3 ab 2.9 a 1.2 b
subsp. macrochele
T. schmiedickeanus 4.7 a 2.2 bc 4.1 ab 0.8 c
subsp. schmiedickeanus
T. schmiedickeanus 9.5 a 4.8 b 10.5 a 1.7 c
subsp. schwarzii
T. subterraneus 13.1 a 10.4 b 10.5 b 8.8 b
T. valdezianus 7.7 c 11.7 ab 13.6 a 8.5 bc
* Treatment means followed by the same letter within a species do not differ significantly at P≤0.05.
Table 1. New shoot generation by areole activation in Turbinicarpus explants taken from shoots conserved by 12 months in
media with mannitol, sorbitol or incubated at low temperature.

without treatments to slow their growth was highly
variable (Fig. 1a−p). The species T. schmiedickeanus
ssp. klinkerianus showed the least relative growth, at
165% in a 60 d incubation period (Fig. 1j), while
T. hoferi stood at the other extreme, with a fresh
weight increase of 3520% over the same time period
(Fig. 1b). The remaining species showed growth rates
within this range. These differences may be a reflec-
tion of their normal rate of growth in natural condi-
tions, but may also result from the possibility that al-
though the MS basal medium is suitable for in vitro
growth in all species, it may not be the optimal me-
dium for all species. Some species of the genus grow
in calcareous and alkaline soils, so they may require
some modifications of the basal medium to reach
their full growth potential.
Both osmotic agents added to the medium, and
incubation at low temperature, reduced in vitro
growth rate in 12 of the 16 taxa (Fig. 1). In some
cases the reduction was remarkable, with the shoots
showing practically no growth after 60 days of in-
cubation. As exceptions to the aforementioned,
the treatment with osmotic agents (mannitol and
sorbitol) did not reduce the growth in T. laui (Fig.
1c), whereas in T. schmiedickeanus subsp. schwar-
zii only mannitol caused this effect (Fig. 1m). In T.
schmiedickeanus subsp. flaviflorus the treatment that
was negative for reducing growth was the incubation
at low temperatures (Fig. 1h). Finally, in T. pseudo-
macrochele subsp. lausseri the growth of shoots in-
cubated in the presence of sorbitol was higher than
the control, while mannitol and incubation at low
temperature did reduce the growth rate (Fig. 1e).
In other plant groups it has been observed that al-
though tissue preservation under slow-growth con-
ditions is generally efficient, there is a loss of tissue
due to death during the storage period. For example,
in the potato, the survival rate of explants after 12
months was between 12.0 and 67.6% with different
mannitol and sorbitol treatments (Gopal 2002). In
56 PÉREZ-MOLPHE-BALCH ET AL—IN VITRO CONSERVATION
this study no shoot died, either after 60 days (when
growth was measured) or after the full 12-month
term of the slow-growth storage (Fig. 2a−c). This in-
dicates that the treatments used to reduce the rates
of growth do not harm the studied cacti. This must
be related to the normal growth kinetics of these
plants, which remain dormant during the dry and
cold periods of the year, and only increase their bio-
mass during warm weather when there is water avail-
ability (Nobel 1994). These favorable or unfavorable
conditions, which determine the growth or lack of it
in cacti in nature, can be easily imitated in in vitro
systems, as has been demonstrated in this work.
As for the ability to regenerate whole plants from
tissue preserved for 12 months under the conditions
described above, it was noted that this property was
not affected by any of the strategies used to reduce
growth in eight of the studied species (Table 1) (Fig.
2d−f ). In three species, only the conservation treat-
ment at low temperatures significantly decreased
their ability to produce shoots, while osmotic agents
did not alter this. In two species, both the low tem-
perature and the mannitol reduced the regeneration
capacity of the tissues. In T. pseudopectinatus, only
mannitol negatively affected shoot production after
12 months of storage. Only in the species T. subterra-
neus was it observed that the three tested treatments
significantly decreased the regeneration capacity of
the tissues, so there is a risk of viability loss involved
in the medium or long term if these treatments are
used to conserve germplasm in this case. In this par-
ticular species other concentrations of osmotic agents
and/or other temperatures should be tested in order
to find a treatment that slows growth without af-
fecting the regeneration capacity. Finally, in T. val-
dezianus an interesting phenomenon was observed;
the tissues that were preserved for 12 months in the
presence of osmotic agents, increased their capacity
for regeneration in comparison to those tissues that
never came in contact with the osmotic agents.
The shoots generated in these experiments rooted
in a 50% diluted MS basal medium, and the plants
thus obtained were transferred to soil. The observed
rooting efficiencies were between 56 and 95%, and
survival rates in soil were between 75 and 95%, co-
inciding with the ranges reported previously for
these species (Dávila-Figueroa et al. 2005). There
were differences between species in terms of their
frequencies of rooting and survival in soil, but there
were no effects within species due to the different
treatments used to slow shoot growth during the
storage stage (data not shown).
We have shown that germplasm conservation sys-
tems based on slow-growth conditions consisting of low
temperature and osmotic agents are effective in cacti of
the genus Turbinicarpus. Of particular interest is the use
of mannitol and sorbitol for this purpose, as they are
relatively inexpensive compounds to use and require no
special equipment or additional expenditure of energy,
in contrast to the use of low temperatures. These treat-
ments will help maintain viable germplasm collections
with a minimum investment of time and resources,
since under normal conditions these species must be
transferred to fresh medium every 60 d, as after that
time the media become exhausted and the plant tissues
die, whereas with the developed protocol described here
the subculture period may be extended to at least 12
months. On the other hand, tissues stored this way can
be retrieved at any time from these slow-growth systems
and transferred to regeneration media in order to obtain
the required number of plants and thus have an unlim-
ited supply of plant material that can be used for various
purposes without resorting to collecting wild plants.
Acknowledgements:
This work was supported by the Fondo Sectorial
de Investigación Ambiental SEMARNAT-CONA-
CYT (SEMARNAT-2002-C01-0057) and the Uni-
versidad Autónoma de Aguascalientes (PIBT-03-3n).
We thank Bernardo Pérez Zamorano for his help in
preparing the manuscript.
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